Adaptive laboratory evolution (ALE) has been used to study and solve pressing questions about evolution, especially for the study of the development of mutations that confer increased fitness during evolutionary processes. In this contribution, we investigated how the evolutionary process conducted with the PTS⁻ mutant of Escherichia coli PB11 in three parallel batch cultures allowed the restoration of rapid growth with glucose as the carbon source. The significant findings showed that genomic sequence analysis of a set of newly evolved mutants isolated from ALE experiments 2–3 developed some essential mutations, which efficiently improved the fast-growing phenotypes throughout different fitness landscapes. Regulator galR was the target of several mutations such as SNPs, partial and total deletions, and insertion of an IS1 element and thus indicated the relevance of a null mutation of this gene in the adaptation of the evolving population of PB11 during the parallel ALE experiments. These mutations resulted in the selection of MglB and GalP as the primary glucose transporters by the evolving population, but further selection of at least a second adaptive mutation was also necessary. We found that mutations in the yfeO, rppH, and rng genes improved the fitness advantage of evolving PTS⁻ mutants and resulted in amplification of leaky activity in Glk for glucose phosphorylation and upregulation of glycolytic and other growth-related genes. Notably, we determined that these mutations appeared and were fixed in the evolving populations between 48 and 72 h of cultivation, which resulted in the selection of fast-growing mutants during one ALE experiments in batch cultures of 80 h duration.

Key points

• ALE experiments selected evolved mutants through different fitness landscapes in which galR was the target of different mutations: SNPs, deletions, and insertion of IS.

• Key mutations in evolving mutants appeared and fixed at 48–72 h of cultivation.

• ALE experiments led to increased understanding of the genetics of cellular adaptation to carbon source limitation.

自适应实验室进化（ALE）已用于研究和解决有关进化的紧迫问题，特别是用于研究在进化过程中赋予适应性的突变的发展。在此贡献中，我们调查与PTS进行的进化过程中如何^-突变体的大肠杆菌在三个平行的分批培养PB11允许快速增长的以葡萄糖为碳源的恢复。重大发现表明，从ALE实验2-3分离出的一组新近进化的突变体的基因组序列分析产生了一些必要的突变，这些突变有效地改善了不同健身环境中快速增长的表型。稳压器galR在平行ALE实验中，SNPs是一些突变的目标，例如SNP，部分和全部缺失以及IS1元件的插入，因此表明该基因无效突变与PB11进化种群的适应有关。这些突变导致正在进化的群体选择MglB和GalP作为主要的葡萄糖转运蛋白，但是还需要进一步选择至少第二个自适应突变。我们发现yfeO，rppH和rng基因中的突变改善了不断发展的PTS的适应性优势^-突变体，并导致Glk的泄漏活性增强，从而导致葡萄糖磷酸化以及糖酵解和其他与生长相关的基因的上调。值得注意的是，我们确定这些突变在48至72 h的不断发展的种群中出现并固定下来，从而在80 h的分批培养中的一次ALE实验中选择了快速生长的突变体。

关键点

• ALE实验通过不同适应度的环境选择了进化突变体，其中galR是不同突变的目标：SNP，缺失和IS插入。

•进化中的突变体中出现了关键突变，并在培养48-72 h内固定下来。

• ALE实验导致人们对细胞适应碳源限制的遗传学有了更多的了解。